Laboratory and clinical procedures involving biospecific affinity reactions have dramatically affected health care and biological research. Such reactions are commonly employed in testing biological samples, such as blood or urine, for the identification, quantification, or both, of a wide range of target substances, for example, particular chemical substances that have been correlated or associated with various disease conditions. Biological entities such as cells, proteins, nucleic acid sequences, e.g., mRNA, and the like are preferred target substances as that term is used herein for utilization of the present invention.
Various methods are available for determining the above-mentioned target substances based upon complex formation or a binding reaction between the substance of interest, i.e., a ligand, and its specific binding partner which preferentially binds to the ligand and not to other constituents that may be present in the sample to be tested. In each instance, the occurrence or degree of target substance/binding partner complex formation is determinable.
Assays typically used in the art include immunoassays, hybridization assays and protein-ligand assays. Immunoassays are based upon the specificity of an antibody for an antigen. Hybridization assays are based on the specificity of complementary nucleic acid sequences, i.e., on the hybridization of nucleic acid probes with target nucleic acids. Protein-ligand assays depend on the affinity of a binding site on a protein for a specific ligand, e.g., streptavidin for biotin.
In each assay types, quantification of the target substance requires a physical separation of bound from free labeled ligand or labeled receptor. Bound/free separations may be accomplished, e.g., gravitationally by settling, or, alternatively, by centrifugation of finely divided particles or beads coupled to the target substance. Such particles or beads can be made magnetic and the bound/free separation step may be accomplished magnetically. Magnetic particles are well known in the art, as is their use in immune and other biospecific affinity reactions. See, for example, U.S. Pat. No. 4,554,088 and Immunoassays for Clinical Chemistry, Hunter et al., eds., Churchill Livingston, Edinborough (1983) pp. 147-162.
Magnetic particles ranging in size from 3 nm to many microns have been described in the patent literature, including, by way of example, U.S. Pat. Nos. 3,970,518; 4,018,886; 4,230,685; 4,267,234; 4,452,773; 4,554,088; 4,659,678; 4,978,610; and 5,200,084. Such small magnetic particles have proved to particularly useful in analyses involving biospecific affinity reactions. They are conveniently coated with biofunctional polymers, such as proteins, provide very high surface areas and give reasonable reaction kinetics.
These small magnetic particles generally fall into two broad categories. The first category includes particles that are permanently magnetized; and the second comprises particles that become magnetic only when subjected to a magnetic field, i.e., they are magnetically responsive particles, for example, paramagnetic particles.
The specific magnetic separation apparatus/method used for bound/free separations of target substance-bearing magnetic particles from test media will depend on the nature and particle size of the magnetic particle. Several magnetic separation devices are commercially available, which can readily remove micron size ferromagnetic particles from solution by employing relatively inexpensive permanent magnets. Examples of such magnetic separators are the MAIA Magnetic Separator manufactured by Serono Diagnostics, Norwell, Mass., U.S.A., the DYNAL MPC-1 manufactured by DYNAL, Inc., Great Neck, N.Y., U.S.A., and the BioMag Separator, manufactured by Advanced Magnetics, Inc., Cambridge, Mass., U.S.A. A similar magnetic separator, manufactured by Ciba-Corning Medical Diagnostics, Wampole, Mass., is provided with rows of bar magnets arranged in parallel and located at the base of the separator. This device accommodates 60 test tubes, with the closed end of each tube fitting into a recess between two of the bar magnets.
Such magnetic separators, however, have the disadvantage that the magnetic capture is accomplished by using positionally-fixed magnets placed external to the wall of the tube of the reaction vessel, and the magnetic particles are collected on the side of the tube. To release the particles, the vessels must be removed from the field of the magnets. This technique is very cumbersome when attempting to separate and process a large number of samples, e.g., ninety-six, as common in multiwell plates such as Microtiter.TM. plates, trademark product of Dynal.
Another approach is manifest in the MACS device made by Miltenyi Biotec GmbH, Gladbach, West Germany, which employs a column filled with a nonrigid steel wool matrix in cooperation with a permanent magnet. In operation, the enhanced magnetic field gradient produced in the vicinity of the steel wool matrix attracts and retains the magnetic particles while the nonmagnetic test medium passes through and is removed from the column. See, R. R. Oder, IEEE Trans. Magnetics, 12 (1976) 428-435; C. Owen and P. Liberti, Cell Separation: Methods and Selected Applications, Vol. 5, Pretlow and Pretlow eds., Academic Press, New York (1986).
It has been found, however, that use of the steel wool matrix of such prior art devices often gives rise to nonspecific entrapment of biological entities other than the target substances which cannot be removed completely without extensive washing and resuspension of the particles bearing the target substance. Moreover, the size of the column in many of the prior art devices requires substantial quantities of experimental materials, which pose an impediment to their use in performing various useful laboratory-scale separations. In addition, the steel wool matrix may be harmful to certain sensitive cell types.
Although the aforementioned prior art devices afford certain advantages in performing medical or biological analyses based on biospecific affinity reactions, the systems developed to date have not been entirely satisfactory for the above-mentioned reasons. Accordingly, it would be desirable to provide an apparatus which is of relatively simple construction and operation, which reduces loss of immobilized target substance and entrapment of nontarget substances, and employs standard multiwell plates and the like, so as to be of practical utility in conducting various laboratory-scale separations, particularly in protein ligand and hybridization assays.